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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Publication from the open literature. No detailed description of the substance tested and on test method (such as incubation conditions). No positive/negative controls included. Cholesterol has been tested in a battery of 135 compounds. All information on genotoxicity will be considered together as weight of evidence for this endpoint.

Data source

Reference
Reference Type:
publication
Title:
Genotoxic activity and potency of 135 compounds in the Ames reversion test and in a bacterial DNA-repair test.
Author:
De Flora S., Zanacchi P., Camoirano A, Bennicelli C., Badolati G.S.
Year:
1984
Bibliographic source:
Mutation Research 133, 161-198

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The plate-incorporation test was performed according to the standard procedure described by Ames et al (1975), with some revisions as published by Maron and Ames (1983).
The DNA-repair test in E. coli was performed similar to the procedure as desribed by Kada et al (1980) in the rec-assay system with B. subtilis and by McCarrol et al (1981) with various strains of E. coli.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Cholesterol
EC Number:
200-353-2
EC Name:
Cholesterol
Cas Number:
57-88-5
Molecular formula:
C27H46O
IUPAC Name:
cholest-5-en-3-ol
Details on test material:
- Name of test material (as cited in study report): Cholesterol
- Analytical purity: reagent pure grade, Sigma

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain
Species / strain / cell type:
other: S. typhimurium TA1535, TA1537, TA1538, TA98, TA100; E. coli WP2, WP67, CM871
Metabolic activation:
with and without
Metabolic activation system:
Prepared according to Ames et al (1975), containing 10% liver S9 fractions from Aroclor-treated Sprague-Dawley rats (protein concentration adjusted to 30 mg/ml).
Test concentrations with justification for top dose:
not specified
Vehicle / solvent:
DMSO
Details on test system and experimental conditions:
The plate-incorporation test was performed according to the standard procedure described by Ames et al (1975), with some revisions as published by Maron and Ames (1983). Each compound (total of 135 compounds, including cholesterol (reagent pure grade, Sigma)) was tested with each strain (5 his- S. typhimurium strains, TA1535, TA1537, TA1538, TA98, TA100; 3 isogenic E. coli strains, WP2 (repair-proficient), WP67 (uvrA-, polA-), CM871 (uvrA-, recA-, lexA-)), both with and without S9 mix at various dilutions (duplicate or triplicate plates) performed by a geometric ratio of 2, starting from its solubility or toxicity limit. Throughout the period of experiments the spontaneous reversion rate of Salmonella tester strains was within the range indicated by Ames et al (1975) and Maron and Ames 91983).
The DNA-repair test in E. coli was performed similar to the procedure as desribed by Kada et al (1980) in the rec-assay system with B. subtilis and by McCarrol et al (1981) with various strains of E. coli. The initial concentration of each compound was governed either by its solubility or by its toxicity for bacteria. Starting from this concentration compounds were further diluted in nutrient broth for a total of 8 2-fold dilutions. In confirmatory assays only 4 dilutions were tested. The assay was performed with and without S9. Per well 1000 bacteria were added. Controls of bacterial density were prepared by seeding 100 µl in nutrient agar pour plates. The exact number of bacteria used was scored after 16h at 37°C. Bacterial growth was visually evaluated after 16h at 37°C. Each compound was assayed in at least 5 separate experiments in order to check the reproducibility of results.
Evaluation criteria:
Plate-incorporation test: criteria for positivity of results included rate of increase of induced versus spontaneous revertants, dose dependence and reproducibility in separate experiments. The mutagenic potency was expressed by dividing the number of revertants in excess of controls by the corresponding amount of compounds (in nmoles). For negative compounds 'less than' figures were calculated by dividing an arbitrary value of 100 revertants by the nmoles corresponding to the maximum dose of compound tested.
DNA-repair test in E. coli: A ratio between the Minimum Inhibitory Concentrations (MICs) in repair-proficient and -deficient strain greater than 2 (confirmed in 5 experiments) was considered to be significant in this test.
Statistics:
not indicated

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA1535, TA1537, TA1538, TA 98, TA100; E. coli WP2, WP67, CM871
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
Positive controls validity:
not examined
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

S. typhimurium reversion test: Potency of cholesterol (revertants/nmole) = <0.01

E. coli repair test: MIC (µg) +/-S9 >1000; potency ( ΔMICs/nmole) = <0.0002

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In a S. typhimurium reversion assay with strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and in an E. coli DNA-repair test with WP2, WP67 and CM871 cholesterol showed no mutagenic.
Executive summary:

The gentoxic activity and potency of 135 compounds, amongst which cholesterol, were tested in the Ames reversion assay and in a bacterial DNA-repair assay. In the Ames assay, cholesterol was tested with S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and in the DNA-repair assay with E. coli strains WP2, WP67 and CM871. The assays were performed with and without metabolic activation (S9) in duplicate or triplicate (Ames test) or in 5 experiments (bacterial DNA-repair test). In these tests, cholesterol did not show genotoxic potential.